Shi Rui, Liu Fei, Huo Guicheng, Yang Lijie
Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Aug;25(8):1240-6.
To evaluate the absolute quantification of a target gene transcription in engineered lactic acid bacteria, we developed the Real-time RT-PCR based on DNA subtraction. We isolated the total RNA from the bacteria samples by glass bead, and then analyzed the Ct data of real-time RT-PCR by DNA subtraction assay. Using this method, we successfully estimated the expression level of CBHII gene in the strain of genetic engineered Lactococcus lactis. Since this method could avoid the mRNA copy number loss, it could be used to estimate the expression of other genes in lactic acid bacteria.
为了评估工程化乳酸菌中目标基因转录的绝对定量，我们开发了基于DNA扣除法的实时逆转录聚合酶链反应。我们通过玻璃珠从细菌样本中分离出总RNA，然后通过DNA扣除法分析实时逆转录聚合酶链反应的Ct数据。使用这种方法，我们成功地估计了基因工程乳酸乳球菌菌株中CBHII基因的表达水平。由于这种方法可以避免mRNA拷贝数的损失，它可用于估计乳酸菌中其他基因的表达。
